Generation of GARP2-Specific Knockout Mice Using Zinc Finger Nuclease Technology

Abstract

 

Purpose

 

The Cngb1 locus encodes the rod photoreceptor cGMP-gated cation channel β-subunit and two soluble glutamic acid rich proteins, GARP1 and GARP2. GARPs are generated by alternative splicing towards the 5’-end of the gene. A knockout null mouse deleted for all three proteins in rods exhibits disorganized rod outer segment (ROS) structure and an attenuated photoresponse. To further understand the role of GARPs in ROS we have used zinc finger nuclease (ZFN) genome editing technology to selectively remove a GARP2 unique exon from the mouse genome.

 

Methods

 

A FokI restriction nuclease site adjacent to suitable zinc finger DNA recognition sequences was identified downstream of GARP2-specific exon 12a. Two ZFN mRNAs encoding zinc finger DNA recognition domains and half of FokI restriction endonuclease were generated by T7 transcription using Sigma CompoZr technology and tested for nuclease activity in cultured mouse Neuro2A cells. ZFN RNAs were co-injected into male pronuclei of C57Bl/6 fertilized embryos and founder mice were screened for deletions by PCR and DNA sequencing.

 

Results

 

Zinc finger nuclease RNA introduced into cultured cells yielded significant target site cleavage of up to 11% of targeted plasmid. Microinjection of 235 embryos produced 14 founder mice of which one, by PCR indicated different deletions on both alleles. DNA sequence analysis of the PCR products demonstrates 826 and 514 bp deletions. The 826 bp deletion covers all of GARP2 specific exon 12a and the 514 bp deletion retains the acceptor splice site and the 8 amino acid protein coding region and stop codon within this exon. This founder mouse was crossed to wild type mice yielding expected Mendelian transmission of each deletion allele establishing germline transmission.

 

Conclusions

 

Two germline transmitting GARP2-specific exon knockout mice have been generated using ZFN technology. One KO allele is a GARP2-specific null that will allow assessment of the requirement for GARP2 function in rods, and the partial KO may be a GARP2 hypomorph with reduced expression of GARP2 that will complement our GARP2 overexpression studies. ZFN technology is a rapid and efficient method of targeted genome editing.

 

 

ZFN-mediated genome editing PCR results. WT mice yield a 2.15 kb amplicon. Crossing of the founder mouse (F0) to WT mice produced offspring that were heterozygous for the WT allele and one of the deletion alleles (Δ826 or Δ514).

 

Keywords: 740 transgenics/knock-outs • 569 ion channels • 688 retina